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The Optical Quality Analysis System (OQAS, Visiometrics) provides objective measurements of image formed onto retina, by combining quantification of ocular media transparency and of optical aberrations. In order to evaluate its contribution in the assessment of age-related cataract, we conducted a monocentric clinical study to determine the relationships between clinical grading of lens opacity, OQAS parameters, and parameters required for cataract surgery by phacoemulsification with ultrasound (called \"phacodynamics\"). Clinical parameters were: best-corrected visual acuity (BCVA, expressed as Log of minimal angle resolution (logMAR)) and the lens opacity classification system III (LOCS III) as a gold standard determined by two independent observers who graded total cataract and nuclear, cortical and posterior sub capsular components. The OQAS provided an objective scatter index (OSI), a modulation transfer function (MTF, expressed in cycle per degree (cpd)) and a Strehl ratio (SR) used as an aberration marker. Patients were operated on by the same surgeon using a phacoemulsification machine that provided the cumulative dissipated energy (CDE) and total ultrasound time (US time) necessary to extract the lens. Patients with poor compliance, corneal or retinal diseases impairing OSI, or who required surgical settings variation, were excluded. Twenty-one eyes of 21 patients aged 76±8 years were analyzed. They were 11 pure nuclear, 3 pure cortical, and 7 mixed cataracts. Mean LOCS III and OSI were respectively: 4.86 ±2.03 and 6.12 ±3.07 (mean±SD). Medians (10°-90° percentiles) were: for BCVA 0.30 (0.10-0.70) logMAR, for MTF cutoff 9.31 (1.54-30.57) cpd, for SR 0.071 (0.042-0.146), for CDE 8.04 (5.74-23.29) and for US time 58 (39-116) seconds. LOCS III was significantly correlated (spearman r, rs) with BCVA (rs = 0.561, p = 0.008), CDE (rs = 0.457, p = 0.038) and US time (rs = 0.647, p = 0.002). The three OQAS parameters significantly correlated (all rs ≥ 0.526, p<0.05) with BCVA, and LOCS III grading, but the strongest correlations were found with OSI for cortical components and with MTF for nuclear components: only OSI may be used objectively to assess the effect of cortical components on optical quality, and MTF cutoff-integrating scattering and aberrations-seems the best objective parameter for clinical assessment of nuclear cataracts. The three OQAS parameters were also significantly correlated (rs) with CDE, and with US time only for pure nuclear cataracts: OSI had the strongest correlations with phacodynamics (rs = 0.693, p = 0.022 with CDE and rs = 0.703, p = 0.019 US time). OSI increased with cortical components not requiring higher CDE. When measured in optimal conditions (good compliance, no retinal or ocular surface or tear film diseases), the three OQAS parameters are complementary for objective grading of cataract. In the future, they may help to optimize surgical parameters, especially energy distribution, in femtosecond laser assisted cataract surgery.

Maintenance of corneal transparency is crucial for vision and depends mainly on the endothelium, a non-proliferative monolayer of cells covering the inner part of the cornea. When endothelial cell density falls below a critical threshold, the barrier and \"pump\" functions of the endothelium are compromised which results in corneal oedema and loss of visual acuity. The conventional treatment for such severe disorder is corneal graft. Unfortunately, there is a worldwide shortage of donor corneas, necessitating amelioration of tissue survival and storage after harvesting. Recently it was reported that the ROCK inhibitor Y-27632 promotes adhesion, inhibits apoptosis, increases the number of proliferating monkey corneal endothelial cells in vitro and enhance corneal endothelial wound healing both in vitro and in vivo in animal models. Using organ culture human cornea (N = 34), the effect of ROCK inhibitor was evaluated in vitro and ex vivo. Toxicity, corneal endothelial cell density, cell proliferation, apoptosis, cell morphometry, adhesion and wound healing process were evaluated by live/dead assay standard cell counting method, EdU labelling, Ki67, Caspase3, Zo-1 and Actin immunostaining. We demonstrated for the first time in human corneal endothelial cells ex vivo and in vitro, that ROCK inhibitor did not induce any toxicity effect and did not alter cell viability. ROCK inhibitor treatment did not induce human corneal endothelial cells proliferation. However, ROCK inhibitor significantly enhanced adhesion and wound healing. The present study shows that the selective ROCK inhibitor Y-27632 has no effect on human corneal endothelial cells proliferative capacities, but alters cellular behaviours. It induces changes in cell shape, increases cell adhesion and enhances wound healing ex vivo and in vitro. Its absence of toxicity, as demonstrated herein, is relevant for its use in human therapy.

The risk of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) transmission through corneal graft is an ongoing debate and leads to strict restrictions in corneas procurement, leading to a major decrease in eye banking activity. The aims of this study are to specifically assess the capacity of human cornea to be infected by SARS-CoV-2 and promote its replication ex vivo, and to evaluate the real-life risk of corneal contamination by detecting SARS-CoV-2 RNA in corneas retrieved in donors diagnosed with Coronavirus Disease 2019 (COVID-19) and nonaffected donors. To assess the capacity of human cornea to be infected by SARS-CoV-2, the expression pattern of SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE-2) and activators TMPRSS2 and Cathepsins B and L in ocular surface tissues from nonaffected donors was explored by immunohistochemistry (n = 10 corneas, 78 ± 11 years, 40% female) and qPCR (n = 5 corneas, 80 ± 12 years, 40% female). Additionally, 5 freshly excised corneas (80 ± 12 years, 40% female) were infected ex vivo with highly concentrated SARS-CoV-2 solution (106 median tissue culture infectious dose (TCID50)/mL). Viral RNA was extracted from tissues and culture media and quantified by reverse transcription quantitative PCR (RT-qPCR) (viral RNA copies) 30 minutes (H0) and 24 hours (H24) after infection. To assess the risk of corneal contamination by SARS-CoV-2, viral RNA was tested by RT-qPCR (Ct value) in both corneas and organ culture media from 14 donors diagnosed with COVID-19 (74 ± 10 years, 29% female) and 26 healthy donors (79 ± 13 years, 57% female), and in organ culture media only from 133 consecutive nonaffected donors from 2 eye banks (73 ± 13 years, 29% female). The expression of receptor and activators was variable among samples at both protein and mRNA level. Based on immunohistochemistry findings, ACE-2 was localized mainly in the most superficial epithelial cells of peripheral cornea, limbus, and conjunctiva, whereas TMPRSS2 was mostly expressed in all layers of bulbar conjunctiva. A significant increase in total and positive strands of IP4 RNA sequence (RdRp viral gene) was observed from 30 minutes to 24 hours postinfection in central cornea (1.1 × 108 [95% CI: 6.4 × 107 to 2.4 × 108] to 3.0 × 109 [1.4 × 109 to 5.3 × 109], p = 0.0039 and 2.2 × 107 [1.4 × 107 to 3.6 × 107] to 5.1 × 107 [2.9 × 107 to 7.5 × 107], p = 0.0117, respectively) and in corneoscleral rim (4.5 × 109 [2.7 × 109 to 9.6 × 109] to 3.9 × 1010 [2.6 × 1010 to 4.4 × 1010], p = 0.0039 and 3.1 × 108 [1.2 × 108 to 5.3 × 108] to 7.8 × 108 [3.9 × 108 to 9.9 × 108], p = 0.0391, respectively). Viral RNA copies in ex vivo corneas were highly variable from one donor to another. Finally, viral RNA was detected in 3 out of 28 corneas (11%) from donors diagnosed with COVID-19. All samples from the 159 nonaffected donors were negative for SARS-CoV-2 RNA. The main limitation of this study relates to the limited sample size, due to limited access to donors diagnosed with COVID-19 and concomitant decrease in the procurement corneas from nonaffected donors. In this study, we observed the expression of SARS-CoV-2 receptors and activators at the human ocular surface and a variable increase in viral RNA copies 24 hours after experimental infection of freshly excised human corneas. We also found viral RNA only in a very limited percentage of donors with positive nasopharyngeal PCR. The low rate of positivity in donors diagnosed with COVID-19 calls into question the utility of donor selection algorithms. Agence de la Biomédecine, PFS-20-011 https://www.agence-biomedecine.fr/.

Keratoconus (KC) is a multifactorial progressive ectatic disorder characterized by local thinning of the cornea, leading to decreased visual acuity due to irregular astigmatism and opacities. Despite the evolution of advanced imaging methods, the exact etiology of KC remains unknown. Our aim was to investigate the involvement of corneal epithelium in the pathophysiology of the disease. Corneal epithelial samples were collected from 23 controls and from 2 cohorts of patients with KC: 22 undergoing corneal crosslinking (early KC) and 6 patients before penetrating keratoplasty (advanced KC). The expression of genes involved in the epidermal terminal differentiation program and of the oxidative stress pathway was assessed by real time PCR analysis. Presence of some of the differentially expressed transcripts was confirmed at protein level using immunofluorescence on controls and advanced KC additional corneal samples. We found statistically significant under-expression in early KC samples of some genes known to be involved in the mechanical resistance of the epidermis ( KRT 16, KRT14 , SPRR1A , SPRR2A , SPRR3 , TGM1 and TGM5 ) and in oxidative stress pathways ( NRF2 , HMOX1 and HMOX2 ), as compared to controls. In advanced KC samples, expression of SPRR2A and HMOX1 was reduced. Decreased expression of keratin (KRT)16 and KRT14 proteins was observed. Moreover, differential localization was noted for involucrin, another protein involved in the epidermis mechanical properties. Finally, we observed an immunofluorescence staining for the active form of NRF2 in control epithelia that was reduced in KC epithelia. These results suggest a defect in the mechanical resistance and the oxidative stress defense possibly mediated via the NRF2 pathway in the corneal keratoconic epithelium.

The cornea, the anterior meniscus-shaped transparent and refractive structure of the eyeball, is the first mechanical barrier of the eye. Its functionality heavily relies on the health of its endothelium, its most posterior layer. The treatment of corneal endothelial cells (CECs) deficiency is allogeneic corneal graft using stored donor corneas. One of the main goals of eye banks is to maintain endothelial cell density (ECD) and endothelial barrier function, critical parameters influencing transplantation outcomes. Unlike in vivo , the stored cornea is not subjected to physiological mechanical stimuli, such as the hydrokinetic pressure of the aqueous humor and intraocular pressure (IOP). YAP (Yes-Associated Protein), a pivotal transcriptional coactivator, is recognized for its ability to sense diverse biomechanical cues and transduce them into specific biological signals, varying for each cell type and mechanical forces. The biomechanical cues that might regulate YAP in human corneal endothelium remain unidentified. Therefore, we investigated the expression and subcellular localization of YAP in the endothelium of corneas stored in organ culture (OC). Our findings demonstrated that CEC morphology, ECD and cell–cell interactions are distinctly and differentially associated with modifications in the expression, subcellular localization and phosphorylation of YAP. Notably, this phosphorylation occurs in the basal region of the primary cilium, which may play central cellular roles in sensing mechanical stimuli. The sustained recruitment of YAP in cellular junctions, nucleus, and cilium under long-term OC conditions strongly indicates its specific role in maintaining CEC homeostasis. Understanding these biophysical influences could aid in identifying molecules that promote homeostasis and enhance the functionality of CECs.

The monolayer of approximately 300,000 human corneal endothelial cells (hCECs) on the posterior surface of the cornea is essential to maintain transparency but is non-self-regenerative. Corneal blindness can currently only be treated by corneal transplantation, hindered by a global donor shortage, highlighting the need for developing tissue and/or cell therapy. The mass production of these advanced therapy medicinal products requires obtaining high-yield, high-quality endothelial cell cultures characterized by hexagonal shape, low size variability, and high endothelial cell density (ECD). Among the usual critical quality attributes which combine the expression of differentiation markers, ECD and cell morphological parameters, the latter are not optimally measured in vitro by conventional image analysis which poorly recognizes adherent cultured cells. We developed a high-performance automated segmentation using Cellpose algorithm and an original analysis method, improving the calculation of classical morphological parameters (coefficient of variation of cell area and hexagonality) and introducing new parameters specific to hCECs culture in vitro. Considering the importance of the extracellular matrix in vivo, and the panel of molecules available for coating cell culture plastics, we used these new tools to perform a comprehensive comparison of 13 molecules (laminins and collagens). We demonstrated their ability to discriminate subtle differences between cultures.

Retinal melanosome/melanolipofuscin-containing cells (MCCs), clinically visible as hyperreflective foci (HRF) and a highly predictive imaging biomarker for the progression of age-related macular degeneration (AMD), are widely believed to be migrating retinal pigment epithelial (RPE) cells. Using human donor tissue, we identify the vast majority of MCCs as melanophages, melanosome/melanolipofuscin-laden mononuclear phagocytes (MPs). Using serial block-face scanning electron microscopy, RPE flatmounts, bone marrow transplantation and in vitro experiments, we show how retinal melanophages form by the transfer of melanosomes from the RPE to subretinal MPs when the “don’t eat me” signal CD47 is blocked. These melanophages give rise to hyperreflective foci in Cd47 −/− -mice in vivo, and are associated with RPE dysmorphia similar to intermediate AMD. Finally, we show that Cd47 expression in human RPE declines with age and in AMD, which likely participates in melanophage formation and RPE decline. Boosting CD47 expression in AMD might protect RPE cells and delay AMD progression.

Fuchs endothelial corneal dystrophy (FECD) is a slowly evolving, bilateral disease of the corneal endothelium, characterized by an abnormal accumulation of extracellular matrix (ECM) in the basement membrane (Descemet’s membrane, DM). This results in the formation of small round excrescences, called guttae, and a progressive disappearance of endothelial cells. In the intermediate stage, the numerous guttae create significant optical aberrations, and in the late stage, the loss of endothelial function leads to permanent corneal edema. The molecular components of guttae have not been fully elucidated. In the current study, we conducted shotgun proteomics of the DMs, including guttae, obtained from patients with FECD and revealed that 32 proteins were expressed only in the FECD-DMs but not in the DMs of control subjects. Subsequent enrichment analyses identified associations with multiple ECM-related pathways. Immunostaining of flat-mounted DMs confirmed that 4 of the top 5 identified proteins (hemoglobin α, SRPX2, tenascin-C, and hemoglobin γδεβ) were expressed in FECD-DMs but not in non-FECD-DMs. Fibrinogen α was strongly expressed in FECD-DMs, but weakly expressed in non-FECD-DMs. We also demonstrated that matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) can display the in situ spatial distribution of biomolecules expressed in the DM, including the guttae.

This study addressed limitations in calcein-AM-based endothelial viability assays, specifically focusing on pre-stripped DMEK grafts. Key challenges included the suboptimal calcein staining and the incompatibility of the viability assay with subsequent immunofluorescence (IF). Using human corneal grafts, we employed two strategies to optimize calcein staining. Firstly, we improved calcein staining in corneal endothelium by adjusting calcein-AM concentration and diluent, resulting in a threefold increase in fluorescence intensity with 4 µM calcein in Opti-MEM compared to the conventional 2 µM calcein in PBS. Secondly, introducing the trypan blue (TB) post-viability assay greatly reduced non-specific fluorescence, enhancing the contrast of calcein staining. This improvement significantly and importantly decreased both inter-operator’s variability and the time required for viability counting. For the subsequent double IF, an extensive wash is recommended on the fixed and permeabilized graft after the viability assay, which was carried out using Hoechst-Calcein (HC) labeling. The simple technical tips outlined in this study are not only effective for pre-stripped DMEK grafts but may also prove beneficial for other types of corneal grafts, such as PK and DSAEK.

In 2013, a clinical trial was initiated to investigate cell therapy for the treatment of corneal endothelial decompensation. Cultivating human corneal endothelial cells (CECs) while maintaining their functional phenotype is challenging; therefore, establishment of a confirmed protocol is pivotal for obtaining approval from regulatory authorities for use of cellular therapy products. In this study, we evaluated organ culture (OC) as a storage method for donor corneas used as a raw material for establishing CEC cultures. OC allows storage of corneal tissue for conventional corneal transplantation at 31–37 °C for up to 5 weeks, whereas storage at 4 °C is limited to 2 weeks. We investigated 20 pairs of corneas: one cornea of each pair was stored in OC and the other in cold storage for one week before CEC culture. In 15/20 cases, the CECs assumed a hexagonal sheet-like monolayer structure and expressed endothelial function-related markers. CECs were also obtained from OC corneas that had been stored for 1 (n = 19) and 2 (n = 7) months. As a further test, CECs were cultivated from 5 OC corneas that had been transported from France to Japan. In all cases, these corneas, even after international transport, generated CECs that formed hexagonal monolayers with clinically applicable and sufficiently high cell densities. In conclusion, the CEC cultures required for endothelial cell therapy can be obtained from OC corneas without changing the standard storage operating procedures of the eye banks.